As for the method for large-scale peptide synthesis, chemical synthesis methods (liquid phase method and solid phase method), enzymatic synthesis methods and biological synthesis methods utilizing recombinant DNA techniques are known. Currently, the enzymatic synthesis methods and biological synthesis methods are employed for the synthesis of long-chain peptides longer than 50 residues, and the chemical synthesis methods and enzymatic synthesis methods are mainly employed for the synthesis of dipeptides.
In the synthesis of dipeptides by the chemical synthesis methods, operations such as introduction and removal of protective groups for functional groups are necessary, and racemates are also formed. The chemical synthesis methods are thus considered to be disadvantageous in respect of cost and efficiency. They are unfavorable also from the viewpoint of environmental hygiene because of the use of large amounts of organic solvents and the like.
As to the synthesis of dipeptides by the enzymatic methods, the following methods are known: a method utilizing reverse reaction of protease (see non-patent publication No. 1); methods utilizing thermostable aminoacyl t-RNA synthetase (see patent publication Nos. 1 to 4); and methods utilizing non-ribosomal peptide synthetase (hereinafter referred to as NRPS) (see non-patent publication Nos. 2 and 3 and patent publication Nos. 5 and 6).
However, the method utilizing reverse reaction of protease requires introduction and removal of protective groups for functional groups of amino acids used as substrates, which causes difficulties in raising the efficiency of peptide-forming reaction and in preventing peptidolytic reaction. The methods utilizing thermostable aminoacyl t-RNA synthetase have the defects that the expression of the enzyme and the prevention of side reactions forming by-products other than the desired products are difficult. The methods utilizing NRPS are inefficient in that the expression of the enzyme by recombinant DNA techniques is difficult because the enzyme molecule is huge, and in that the supply of coenzyme 4′-phosphopantetheine is necessary.
On the other hand, there exist a group of peptide synthetases that have enzyme molecular weight lower than that of NRPS and do not require coenzyme 4′-phosphopantetheine; for example, γ-glutamylcysteine synthetase, glutathione synthetase, D-alanine-D-alanine (D-Ala-D-Ala) ligase, and poly-γ-glutamate synthetase. Most of these enzymes utilize D-amino acids as substrates or catalyze peptide bond formation at the γ-carboxyl group. Because of such properties, they can not be used for the synthesis of dipeptides by peptide bond formation at the α-carboxyl group of L-amino acid.
The only known example of an enzyme capable of forming a dipeptide by the activity to form a peptide bond at the α-carboxyl group of L-amino acid is bacilysin (dipeptide antibiotic derived from a microorganism belonging to the genus Bacillus) synthetase. Bacilysin synthetase is known to have the activity to synthesize bacilysin [L-alanyl-L-anticapsin (L-Ala-L-anticapsin)] and L-alanyl-L-alanine (L-Ala-L-Ala) (see non-patent publication Nos. 4 and 5). Recently, it has been reported that this enzyme has the activity to form various kinds of dipeptides from various combinations of the same or different free amino acids (see patent publication No. 7).
However, there exists a need for a novel dipeptide-synthesizing enzyme which has substrate specificity different from that of the above enzyme, because the above enzyme can not form all dipeptides efficiently due to its substrate specificity.
The nucleotide sequence of the chromosomal DNA and the presumed nucleotide sequences of genes of Ralstonia solanacearum GMI1000 are both known. However, neither the function of a protein encoded by RSP1486 gene nor whether RSP1486 gene actually encodes a protein having a function is not known.
Patent publication No. 1:
                Japanese Published Unexamined Patent Application No. 146539/83Patent publication No. 2:        Japanese Published Unexamined Patent Application No. 209991/83Patent publication No. 3:        Japanese Published Unexamined Patent Application No. 209992/83Patent publication No. 4:        Japanese Published Unexamined Patent Application No. 106298/84Patent publication No. 5:        U.S. Pat. No. 5,795,738Patent publication No. 6:        U.S. Pat. No. 5,652,116Patent publication No. 7:        WO04/058960 pamphletNon-patent publication No. 1:        J. Biol. Chem., 119, 707-720 (1937)Non-patent publication No. 2:        Chem. Biol., 7, 373-384 (2000)Non-patent publication No. 3:        FEBS Lett., 498, 42-45 (2001)Non-patent publication No. 4:        J. Ind. Microbiol., 2, 201-208 (1987)Non-patent publication No. 5:        Enzyme. Microbial. Technol., 29, 400-406 (2001)        